REPRODUCTIONRESEARCH

Notch signaling represses GATA4-induced expression of involved in steroid biosynthesis

Rajani M George†, Katherine L Hahn‡, Alan Rawls, Robert S Viger1,2 and Jeanne Wilson-Rawls School of Life Sciences, Arizona State University, PO Box 874501, Tempe, Arizona 85827-45012, USA, 1Reproduction, Mother and Child Health, Centre de Recherche du CHU de Que´bec and Centre de Recherche en Biologie de la Reproduction (CRBR), Quebec City, Quebec, Canada and 2Department of Obstetrics, Gynecology, and Reproduction, Laval University, Quebec City, Quebec, Canada G1K 7P4 Correspondence should be addressed to J Wilson-Rawls; Email: [email protected]

†R M George is now at GE Healthcare Life Sciences, 7th Floor Brigade Metropolis, Whitefield, Bangalore 560048, India ‡K L Hahn is now at STEMCELL, Ste. 400, 570 West Seventh Avenue, Vancouver, British Columbia, Canada V5Z 1B3

Abstract

Notch2 and Notch3 and genes of the Notch signaling network are dynamically expressed in developing follicles, where they are essential for granulosa cell proliferation and meiotic maturation. Notch receptors, ligands, and downstream effector genes are also expressed in testicular Leydig cells, predicting a potential role in regulating steroidogenesis. In this study, we sought to determine if Notch signaling in small follicles regulates the proliferation response of granulosa cells to FSH and represses the up-regulation steroidogenic expression that occurs in response to FSH as the follicle grows. Inhibition of Notch signaling in small preantral follicles led to the up-regulation of the expression of genes in the steroid biosynthetic pathway. Similarly, progesterone secretion by MA-10 Leydig cells was significantly inhibited by constitutively active Notch. Together, these data indicated that Notch signaling inhibits steroidogenesis. GATA4 has been shown to be a positive regulator of steroidogenic genes, including STAR protein, P450 aromatase, and 3B-hydroxysteroid dehydrogenase. We observed that Notch downstream effectors HEY1, HEY2, and HEYL are able to differentially regulate these GATA4-dependent promoters. These data are supported by the presence of HEY/HES binding sites in these promoters. These studies indicate that Notch signaling has a role in the complex regulation of the steroidogenic pathway. Reproduction (2015) 150 383–394

Introduction cells have active Notch signaling (Johnson et al. 2001, Hahn et al. 2005, Vanorny et al. 2014), and thus, this It is well established that gonadotropins participate in pathway may mediate the proliferation response of complex feedback loops required for spermatogenesis granulosa cells to FSH in growing follicles by suppres- and folliculogenesis. Follicle-stimulating hormone (FSH) sing their differentiation and the expression of genes is important for the development of spermatogonia and steroidogenesis in Leydig cells. FSH also regulates involved in steroidogenesis. the number of Sertoli and Leydig cells (Baker & The evolutionarily conserved Notch signaling O’Shaughnessy 2001, Baker et al. 2003). During pathway has been shown to be required for follicle folliculogenesis, granulosa cells respond differently to development and fertility. We, and others, have FSH depending on follicle size. In small preantral demonstrated that Notch2, Notch3, and the ligands follicles, FSH induces granulosa cell proliferation. In Jagged1 (Jag1) and Jag2 are expressed dynamically in the larger follicles, however, FSH induces these cells to cells of growing follicles (Johnson et al.2001, express the luteinizing hormone and enzymes Vorontchikhina et al. 2005, Vanorny et al. 2014). The of the steroid biosynthesis pathway, such as P450 downstream effector genes Hes1, Hes5, Hey1, Hey2, aromatase (Cyp19a1), P450 side chain cleavage enzyme and HeyL overlap with Notch and its ligands in the (Cyp11a1), and STAR protein (Star)(Cortvrindt et al. granulosa cells (Johnson et al. 2001, Hahn et al. 2005). 1997, Robker & Richards 1998, Spears et al. 1998, Several lines of evidence indicate that Notch signaling in Kreeger et al. 2005, Kwintkiewicz et al. 2007). The the is necessary for granulosa cell proliferation and mechanism(s) by which small follicle granulosa cells are normal follicle development. In mice deficient for responsive to only the mitogenic activity of FSH remain lunatic fringe (Lfng), a modifier of the extracellular poorly understood. In preantral follicles, all granulosa domain of the Notch receptors, Notch signaling was

q 2015 Society for Reproduction and Fertility DOI: 10.1530/REP-15-0226 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/25/2021 11:48:09AM via free access 384 R M George and others inhibited in granulosa cells, and these contained proteins have also been demonstrated to form complexes aberrant multi-oocyte follicles (MOFs) that did not with GATA factors and repress expression of genes such undergo meiotic maturation properly (Hahn et al. as atrial naturietic factor, myosin heavy chain, and 2005). In ex vivo ovary culture, the addition of Mu¨llerian inhibiting substance through GATA sites g-secretase inhibitors that block Notch signaling resulted (Elagib et al. 2004, Kathiriya et al. 2004, Fischer et al. in a loss of granulosa cell proliferation (Zhang et al. 2011). 2005, Ishiko et al. 2005, Martin et al. 2005, Shirvani Notch2 is necessary for primordial follicle formation et al. 2006, Xiang et al. 2006). (Trombly et al. 2008)andNotch2 deficient ovaries had Transcription of genes in the steroid biosynthesis MOFs that demonstrated a lack of granulosa cell pathway is positively regulated in the gonads by proliferation (Xu & Gridley 2013, Vanorny et al. 2014). (referred to as SF1, gene symbol A similar ovarian phenotype was found in mice with an Nr5a1), a and CREB/CREM (Ito et al. oocyte-specific mutation of Jag1 (Vanorny et al. 2014). 1997, Gurates et al.2003, Ragazzon et al.2006, Notch signaling pathway genes are also expressed in Schimmer & White 2010). In contrast, dosage-sensitive the developing testis and juvenile and adult Leydig cells sex reversal adrenal hypoplasia critical region on (Dirami et al. 2001, von Scho¨nfeldt et al. 2004, Lupien X (NrOb1 gene symbol, referred to as et al. 2006, Tang et al. 2008, Defalco et al. 2013). During Dax1) is a nuclear receptor that acts as a pan gonadogenesis, Notch signaling regulates the differen- transcriptional inhibitor of steroid biosynthetic genes tiation of Leydig cells from progenitors (Tang et al. 2008). (Tremblay & Viger 2001a, Wang et al. 2001, Lalli & Inhibiting Notch signaling increased the number of Sassone-Corsi 2003, Jo & Stocco 2004, Manna et al. Leydig cells, and conversely, expression of constitutively 2009). Notch signaling could regulate steroid biosyn- active Notch resulted in a loss of these cells pre- and thesis by acting on these regulators of steroidogenesis, postnatally (Tang et al. 2008, Defalco et al. 2013). either by inhibiting the transcription of SF1 or by Interestingly, gain- or loss-of-function Notch mutations up-regulating expression of Dax1. resulted in both aberrant testis cord formation and loss of GATA4, a zinc finger domain , has germ cells (Tang et al. 2008). a double zinc finger-binding domain that recognizes the There are four Notch receptors (Notch1–4)in GATA motif in promoters (review Tevosian (2014)). mammals and five ligands: Deltalike1 (Dll1), Dll3, GATA4 is expressed in both Leydig and granulosa cells Dll4, Jag1,andJag2 (Kopan & Ilagan 2009). On ligand (Heikinheimo et al. 1997, Viger et al. 1998, 2004, activation, the Notch intracellular domain (NOTCHICD) Laitinen et al. 2000, Tremblay & Viger 2001a,b, translocates to the nucleus and binds DNA in a complex Martin et al. 2005, Padua et al. 2014, 2015) and is a with an obligate cofactor, RBPJK (Jarriault et al. 1995, Lu key positive transcriptional regulator of genes involved & Lux 1996, Ong et al. 2006), activating the transcription in steroid biosynthesis, including SF1, Cyp19a1, Star, of its downstream target effectors, the HES and HEY proteins. The HEY and HES proteins are two families of 3B hydroxysteroid dehydrogenase (Hsd3b1), and basic helix–loop–helix Orange (bHLH-O) transcriptional Cyp11A1 (Wooten-Kee & Clark 2000, Tremblay & repressors (Nakagawa et al. 2000, Iso et al. 2003, Viger 2001a,b, Tremblay et al. 2002, Bouchard et al. Kokubo et al. 2005). In this way, Notch signaling can 2005, Martin et al. 2005, Bergeron et al. 2015, both activate and repress gene transcription. Schrade et al.2015). Notch signaling has been The HES and HEY proteins are able to repress demonstrated to inhibit GATA factor-mediated transacti- transcription through several distinct mechanisms. They vation of cardiac, hematopoietic, and skeletal muscle can form homodimers that bind to E (CANNTG) or specific promoters (Kathiriya et al. 2004, Fischer et al. N (CACNAG) boxes in target promoters and then recruit 2005, Ishiko et al. 2005, Shirvani et al. 2006), indicating transcriptional repressor complexes (Sasai et al. 1992, that the steroidogenic promoters could also be targets of Kageyama & Nakanishi 1997, Takata & Ishikawa 2003, the Notch pathway. Kokubo et al. 2005, Holderfield & Hughes 2008, Niwa In the present study, we sought to examine the role of et al. 2011). Alternatively, they can repress transcription Notch and its HEY downstream effector proteins in the through the formation of heterodimers with ubiquitously regulation of the expression of genes involved in gonadal expressed class A bHLH factors, such as E12 and E47. steroidogenesis. Our data indicates that Notch signaling The sequestration of these factors in nonfunctional inhibits FSH-induced steroidogenic heterodimers prevents the formation of heterodimers in small follicles. Also, activated Notch blocked the with tissue-specific bHLH activators, such as MYOD synthesis of progesterone in cultured MA-10 Leydig or PARAXIS (Leimeister et al. 2000, Iso et al. 2003). cells. A number of genes encoding important enzymes The HEY proteins also form complexes with non-bHLH involved in the gonadal steroidogenesis pathway are transcription factors and repress their activity. For positively regulated by GATA4 through direct transcrip- example, HEY1 binds to the (Belandia tional activation of the corresponding promoter et al. 2005), and HEY2 binds to the aryl hydrocarbon sequences. We now show that GATA4-dependent receptor nuclear translocator (Chin et al. 2000). The HEY activation of these promoters is also a target for

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Notch-mediated repression, further supporting a role for t-butyl ester (DAPT), a Notch inhibitor (Sigma–Aldrich). Each Notch signaling in the control of steroidogenesis. treatment well received 20 follicles and each treatment was done in duplicate. After 48 h, total follicle RNA was purified for analysis by quantitative RT-PCR (qRT-PCR). The data are the Materials and methods results of three experiments. Cells MA-10 cells, a Leydig tumor cell line (a kind gift from Dr Mario Enzyme immunoassay progesterone assay Ascoli, University of Iowa), were cultured in RPMI-1640 (Life MA-10 cells were seeded at 1!105 cells/well and cultured as Technologies) with 15% horse serum (GE Healthcare Hyclone, above. These cells were transfected using Fugene6 (Invitrogen) Logan, UT, USA), 20 mM HEPES (Sigma–Aldrich), 4 mM at a 6:2 ratio with a maximum of 2 mg of DNA. Empty vector glutamine (GE Healthcare Hyclone), and 50 mg/ml gentamycin was used to equalize total DNA concentration across all (Mediatech, Herndon, VA, USA) on gelatinized plates as transfections. At 48 h post-transfection the cells were trans- described in Mizutani et al.(2006). NIH3T3 cells were ferred into serum-free medium and treated with 1 mM dibutyryl purchased from ATCC (Manassas, VA, USA) and cultured in cAMP (dbcAMP; Sigma–Aldrich) or vehicle for 6 h. The culture DMEM (Life Technologies) with 10% newborn calf serum medium was collected for enzyme immunoassay (EIA). The (Atlanta Biologicals, Flowery Branch, GA, USA) and Primocin concentration of progesterone in the collected medium was (Invivogen, San Diego, CA, USA). determined using an EIA assay per the manufacturer’s protocol (Cayman Chemicals, Ann Arbor, MI, USA). Cells were then Plasmids lysed and RNA was isolated for qRT-PCR. The GATA4 expression plasmid and promoter–luciferase constructs, mouse Star (K902 to C17), Cyp19a1 (K218 to Quantitative RT-PCR C K C 44), and human HSD3B2 ( 224 to 53), have been MA-10 cells were cultured, transfected, and treated with described elsewhere (Tremblay & Viger 2001a,b, Tremblay dbcAMP, as described above. MA-10 cells or intact follicles C et al. 2002, Martin et al. 2005). The pCS2 -Notch1ICD and - were lysed in TRIzol (Invitrogen) for RNA isolation, per the Notch3ICD constructs were kind gifts of Dr Raphael Kopan manufacturer’s protocols. For these studies, three biological (Washington University, St Louis, MO, USA). The mouse replicate experiments were performed. RNA was treated with Notch2ICD (a kind gift of Dr Kathy Loomes, Children’s Hospital DNase I and quantified by NanoDrop prior to cDNA synthesis of Philadelphia, Philadelphia, PA, USA) was cloned into the using SuperScriptIII reverse transcriptase (Invitrogen). For each C pCS2 backbone. The Hey2 expression plasmid and deletion sample, 1 mg of RNA was used for cDNA synthesis. The cDNA constructs Hey2-62 and Hey2-121 were cloned into the was quantified using transcript-specific, intron-spanning C pCS2 vector and confirmed by sequencing. primers and real-time PCR with Syber Green (Eurogentec, Fremont, CA, USA) on an ABI 7900 HT thermocycler using a Animals 384-well format in 10 ml reactions. Products from each primer set were sequenced and analyzed by BLAST (National Center CD1 mice (Mus musculus) were bred and housed in a vivarium for Biotechnology Information (NCBI)) to verify their identity. at Arizona State University (ASU) on a 10 h light:14 h darkness Primer efficiency was determined using a standard curve. For schedule with access to food and water ad libitum. ASU is each transcript, three biological replicates were assayed in accredited by the Association for Assessment and Accreditation triplicate. All samples were normalized to the Gapdh transcript of Laboratory Animal Care (AALAC). All procedures were and relative gene expression was calculated using DDCq carried out in compliance with the ASU Institutional Animal analysis (Haimes & Kelley 2010). For follicle RNA, the control Care and Use Committee and AALAC under an approved sample was follicles cultured with FSH and vehicle. For MA-10 research protocol. cells, the control sample was cells cultured without any treatment. Primer sequences for mouse genes are as follows: Gapdh,50-GGGAAGCCCATCACCATCTT-30 and 50-GCCC- Follicle culture 0 0 TTCTCCATGGTGGTGAA-3 ; Dax1,5-GCCCTTTTCCTGCT- Ovaries from 14-day-old CD1 mice were harvested and follicles GAGATTC-30 and 50-TCACAGCTTTGCACAGAGCAT-30; were mechanically isolated using a fine needle. Small preantral SF1,50-CGCAACAACCTTCTCATTGAGA-30 and 50-TGGA- follicles that were w200 mm in diameter and had attached theca TCCCTAATGCAAGGAGTCT-30; Star,50-AAGCTGTGTGCTG- cells were chosen for culture, as described previously (Murray et al. GAAGCTCCTAT-30 and 50-TGCTTCCAGTTGAGAACCA 1998, Lenie et al. 2004). The follicles were cultured in a-MEM AGCAG-30; Hsd3b1 (mouse homolog of human HSD3B2), (Mediatech) with 5% FBS (Atlanta Biologicals), insulin, transferrin 50-ACACAAGGAAGGAATTCTCCAAGCTG-30 and 50-CCTCC- and selenium (ITS) (Sigma–Aldrich), 50 mg/ml ascorbic acid AATAGGTTCTGGGTACCTT-30; Cyp19a1,50-CAGCAAGT- (Sigma–Aldrich), 0.3% BSA (Sigma–Aldrich), 2 mM glutamine CCTCAAGCATGTTCCA-30 and 50-TTCCACCATTCGAAC (GE Healthcare HyClone), and penicillin/streptomycin (Media- AAGACCAGG-30; Hes1,50-CAACACGACACCGGACAAAC- tech) in the presence of 200 mIU/ml FSH (Sigma–Aldrich) under CAAA-30 and 50-TGGAATGCCGGGAGCTATCTTTCT-30; and one of the following conditions: DMSO vehicle only, 25 or Notch2,50-TGCTGTGGCTCTGGCTGT-30 and 50-TGTGG- 0 50 mM N-(N-(3,5-difluorophenacetyl-L-alanyl))-(S)-phenylglycine TAGGTAACACAGGTCCCT-3 . www.reproduction-online.org Reproduction (2015) 150 383–394

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Luciferase assays significance was determined using one-way ANOVA except for qRT-PCR that was analyzed using two-way ANOVA, NIH3T3 cells were plated at a density of 8!104 cells/ml in P!0.05 for statistical significance at the 95% confidence limit. 24-well plates and cultured as described above. Cells were transfected the next day with Lipofectamine and Plus reagent following the manufacturer’s protocol (Invitrogen). Each transfected well received 0.05 mg of GATA4 expression vector, Results 0.2 mg of steroidogenic gene promoter–luciferase reporter construct, and 0.025 mg of plasmids expressing NotchICDs or FSH induces both proliferation and expression of Hey proteins. All transfections included 0.05 mg of pCMV– steroidogenic genes in granulosa cells depending on eGFP (Invitrogen). Total plasmid DNA was kept at 0.4 mg/well follicle size (Robker & Richards 1998, Kwintkiewicz in all transfections with the addition of empty vector. All assays et al. 2007). Notch2 and Notch3 are expressed in all were done in triplicate on the same plate. The control sample granulosa cells of small growing follicles (Johnson et al. for these assays was transfection of the reporter only. At 48 h 2001), and Notch signaling induces proliferation of post-transfection, cells were lysed in Luciferase Cell Culture many cell types, including granulosa cells (Zhang et al. Lysis Buffer (Promega). Luciferase activity was measured for 2011, Vanorny et al. 2014), thus, it is possible that Notch each well by reacting 20 ml of cell lysate with 100 mlof acts downstream of FSH. To examine this, we cultured Luciferase Assay Buffer (Promega) in 96-well plates, using an preantral follicles from 14-day-old CD1 mice in the FLx800 microplate reader (Biotek Instruments, Winooski, presence or absence of a Notch inhibitor, DAPT, that VT, USA). All samples were normalized to GFP expression. blocks ligand-mediated activation of Notch and relieves All data are the meanGS.D. of three experiments done in triplicate, and statistical significance was determined using a Notch-mediated repression of gene expression (Fig. 1). one-way ANOVA. Follicles were cultured in the presence of FSH under one of the following conditions: DMSO vehicle only or 25 or 50 mM DAPT. After 48 h, total follicle RNA was purified Bioinformatics techniques and gene expression was analyzed using qRT-PCR. Steroid enzyme promoter–luciferase constructs were Hes1, whose transcription is up-regulated by active sequenced and analyzed for the presence of transcription Notch signaling, was significantly inhibited by DAPT factor binding sites using the TFsearch database (Heinemeyer indicating that Notch signaling was blocked (Fig. 1). et al. 1998), which can be found at http://www.cbrc.jp/ Conversely, Notch2 expression was increased through a cbrc-databases. reciprocal signaling pathway, as expected (Vanorny et al. 2014). Genes encoding enzymes important for the synthesis of gonadal steroids – Star, Cyp19a1,and Statistical analyses Hsd3b1 (Payne & Hales 2004) – were significantly All data are the meanGS.D. of a minimum of triplicate up-regulated in follicles cultured in 25 or 50 mM DAPT replicates from three biological experiments. Statistical (Fig. 1). These data indicate that Notch signaling inhibits

A 1.4 Hes110 Notch2

+DAPT 1.2 8 * Notch on Notch off HES/HEY on Transcriptional activators 1.0 0.8 6 * 0.6 4 Target genes off Target genes on 0.4 2 0.2 Relative gene expression Relative gene expression 0.0 * * 0 Mock 25 µM 50 µM Mock 25 µM 50 µM

Hsd3b1 Star Cyp19a1 10 45 45 ** ** 9 * 40 40 * * 8 35 * 35 7 30 30 6 25 25 5 * 20 4 20 3 15 15 2 10 10

Relative gene expression 5 5 Relative gene expression 1 Relative gene expression 0 0 0 Mock 25 µM 50 µM Mock 25 µM 50 µM Mock 25 µM 50 µM

Figure 1 Notch signaling inhibits expression of steroidogenic genes in granulosa cells from preantral follicles. (A) Cartoon describing the assay. Preantral follicles were isolated from 14-day-old CD1 mice and 20 follicles were cultured in the presence of FSH and 0, 25, or 50 mM DAPT to block Notch signaling for 48 h. Gene-specific qRT-PCR was done on total follicle RNA. Hes1 transcription is repressed and demonstrates that DAPT inhibited Notch signaling. Notch2 transcription was increased, as expected. Hsd3b1, Star, and Cyp19a1 demonstrated increased transcription when cultured with FSH and DAPT. Significance was determined by ANOVA, P!0.05, *indicates that the sample is statistically different from the mock sample and **indicates 25 and 50 mm DAPT samples are statistically different from each other. Data are the results of three experiments, each done in triplicate.

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140000 activated Notch receptor did not block or attenuate this ** 120000 effect. Similarly, neither dbcAMP nor activated Notch 100000 * receptors had any effect on the expression of SF1 mRNA 80000 (Fig. 3). These data suggested that Notch may regulate 60000 * the promoters of specific genes necessary for steroid 40000 biosynthesis because progesterone synthesis was inhib-

Progesterone (pg/ml) 20000 ited (Fig. 2). 0 Whether Notch could mediate repression of specific

MP MP CD MP steroid biosynthesis genes was tested using the well- vector otch1ICD otch2I otch3ICD characterized promoters of Star, HSD3B2, and Cyp19a1 N N N Empty CD+dbcA CD+dbcAMP CD+dbcA PII, which are active in granulosa cells and Leydig cells.

otch1I otch2I otch3I These promoters are synergistically activated by GATA4 N N N Empty vector+dbcA and SF1 (Tremblay & Viger 2001a,b, Bouchard et al. Figure 2 Activated Notch inhibits progesterone secretion in MA-10 2005, Martin et al. 2005, Kwintkiewicz et al. 2007, cells. MA-10 cells were transfected with a Notch1–3ICD or empty vector. At 48 h post-transfection, cells were stimulated with dbcAMP to induce steroidogenesis and progesterone was detected in cell 8 supernatants by EIA. Cells transfected with empty vector and treated SF1 with dbcAMP secreted a significant amount of progesterone compared 7 to empty vector transfected cells (**P!0.001). When transfected with 6 Notch1ICD or Notch2ICD, progesterone secretion by dbcAM-treated 5 cells was significantly inhibited, as compared to dbcAMP-treated cells that received empty vector (*P!0.05). Notch3ICD decreased pro- 4 gesterone secretion in the presence of dbcAMP, but not significantly. 3 Transfection with Notch1–3ICD without dbcAMP treatment had no G 2 effect on progesterone synthesis. Data are the means S.E.M. of three Relative expression experiments done in triplicate. 1 0

FSH-induced expression of steroidogenic genes in granulosa cells of small preantral follicles. Notch1ICD Notch2ICD Notch3ICD Empty vector Because Notch pathway genes are also expressed in

Leydig cells, the effect of Notch signaling on steroido- dbcAMP+Notch1ICDdbcAMP+Notch2ICDdbcAMP+Notch3ICD genesis was examined in MA-10 cells, a well-charac- Empty vector+dbcAMP terized Leydig tumor cell line that synthesizes progesterone when stimulated by hormones or dbcAMP 8 Dax1 (Ascoli 1981). MA-10 cells were transfected with the 7 constitutively active intracellular domains of Notch1, 6 Notch2,orNotch3. At 48 h post-transfection the cells 5 were either stimulated with dbcAMP or vehicle and the 4 medium was assayed for progesterone by EIA. Treatment 3 with dbcAMP induced a O30-fold increase in pro- 2

gesterone secretion in untransfected cells (Fig. 2). Relative expression 1 * A combination of dbcAMP treatment and transfection 0 *** of Notch1ICD or Notch2ICD resulted in a significant reduction of progesterone synthesis (*P!0.05; Fig. 2).

Notch3ICD followed this trend but was not statistically Notch1ICD Notch2ICD Notch3ICD significant. Therefore, Notch signaling can inhibit Empty vector synthesis of progesterone in MA-10 Leydig cells. dbcAMP+Notch1ICDdbcAMP+Notch2ICDdbcAMP+Notch3ICD Since Dax1 and SF1 are central regulators of steroido- Empty vector+dbcAMP genesis, we next determined if their expression was Figure 3 Activated Notch does not inhibit Dax1 or SF1 transcription. activated or inhibited, respectively, by Notch signaling. MA-10 cells were transfected with plasmids expressing Notch1–3ICD MA-10 cells were transfected with plasmids expressing or empty vector and cultured in the presence or absence of dbcAMP, NOTCH1–3ICD and induced with dbcAMP for 6 h, and and total RNA was harvested for qRT-PCR using gene specific primers. RNA was isolated for qRT-PCR. As shown in Fig. 3, Dax1 Dax1 transcripts are decreased by dbcAMP but not by activated Notch. SF1 transcription was not affected by either dbcAMP or activated mRNA levels decrease in response to dbcAMP, consistent Notch. Data presented are the relative expression of three with the previously reported decrease in protein (Jo & experiments done in triplicate GS.D.*P!0.05 as compared to Stocco 2004, Manna et al. 2009). Transfection with any untreated MA-10 cells. www.reproduction-online.org Reproduction (2015) 150 383–394

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Schrade et al. 2015, Bergeron et al. 2015). Interestingly, A GATA GATA4 activity is negatively regulated by HEY2 in the Star Luc heart and developing cardiovascular system, making it a 1600 possible target for Notch signaling in gonadal cells also 1400 (Kathiriya et al. 2004, Kokubo et al. 2005). NIH3T3 cells 1200 were used in these studies to reduce background signals 1000 due to endogenous GATA4, SF1, and DAX1 activity. 800 Cells that were transfected with a plasmid expressing * * 600 Gata4 and a Star promoter–luciferase reporter gene 400 * demonstrated greater than sixfold up-regulation of Relative luciferase activity 200 luciferase activity over the reporter alone. This activity 0 was significantly inhibited by co-transfection with Star-luc + + + + + NOTCH1ICD, NOTCH2ICD, or NOTCH3ICD (Fig. 4A). GATA4 – + + + + Similarly, both the K222 to C55 HSD3B2- and the N1ICD – – + – – K284 to K23 Cyp19a1 PII-luciferase reporters were N2ICD – – – + – up-regulated 2.2- and fourfold, respectively, by GATA4. N3ICD – – – – + NOTCH1ICD, NOTCH2ICD, and NOTCH3ICD signi- B GATA ficantly inhibited the ability of GATA4 to activate these HSD3B2 Luc promoters (Fig. 4B and C). These observations are 1000 consistent with the MA-10 cell data and indicate that 900 activated Notch can repress the promoters of specific 800 700 genes in this pathway. 600 * Direct transcriptional repression via Notch signaling 500 is mediated by the HES and HEY bHLH-O repressor 400 * * proteins binding to N and E boxes in the promoters of 300

Relative luciferase activity 200 genes (Iso et al. 2003, Grogan et al. 2008, Heisig et al. 100 2012, Leal et al. 2012). Using TFSearch, an algorithm 0 HSD3B2-luc + + + + + that searches DNA sequences for enhancer binding sites GATA4 – + + + + (Heinemeyer et al. 1998), the promoter regions of Star, N1ICD – – + – – HSD3B2, and Cyp19a1 PII were analyzed for N boxes N2ICD – – – + – (CACNAG consensus) and E boxes (CANNTG consensus) N3ICD – – – – + that could potentially bind HEY family members. The Cyp19a1 PII promoter has two E boxes (Fig. 5). C GATA The HSD3B2 promoter has a single N box, CACAAG, but Cyp19a1 Luc no E boxes. Finally, the Star promoter has an N box and 700 three E boxes (Fig. 5). The GATA sites have been 600 confirmed as GATA4-binding sites that activate tran- 500 scription of these genes in other studies (Tremblay & 400 * * *

Viger 2001a,b, Bouchard et al. 2005, Martin et al. 2005). 300 To determine if the HEY bHLH-O repressors could 200

inhibit GATA4, transcriptional activation of the steroi- Relative luciferase activity dogenesis gene promoters was tested using a similar 100 0 approach to the above discussion. As can be seen in Cyp19a1-luc + + + + + Fig. 6A, GATA4 highly induced Star-luciferase activity GATA4 – + + + + when transfected into 3T3 cells, and this activity was N1ICD – – + – – significantly inhibited by co-transfection with any of the N2ICD – – – + – three HEY proteins. Similarly, the HSD3B2 reporter N3ICD – – – – + O demonstrated a 11-fold increase in luciferase Figure 4 Activated Notch represses GATA4-mediated transcription of activity when co-transfected with GATA4 but was steroidogenic enzyme gene promoters. (A, B and C) NIH3T3 cells were significantly repressed by HEY1 and HEYL only. HEY2 transfected with a specific luciferase reporter gene construct, an had no effect on the HSD3B2 promoter. Co-transfection expression plasmid for GATA4, and a specific activated Notch receptor, of any of the three HEY proteins with the Cyp19a1 as indicated. After 48 h in culture, cells were lysed and luciferase PII-luciferase reporter gene and GATA4 showed activity was determined. GATA4 binds to the promoter regions of the Star, HSD3B2, and Cyp19a1 reporter constructs, inducing transcrip- decreased activity, but only HEY1 inhibited this tion. The GATA4-induced luciferase activity of each gene was inhibited promoter significantly (Fig. 6C). significantly by co-transfection of Notch1–3ICD (*P!0.05). All data HEY proteins also can form complexes with the GATA are the results of three experiments done in triplicate GS.D. Schematics factors and repress transcription. To determine if HEY2 above the graphs depict the reporter constructs.

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Figure 5 TFSearch analysis of steroidogenic gene promoters. The sequence of the promoters in each luciferase reporter gene is presented. Each promoter was examined using TFsearch for the presence of N and E boxes. All of the GATA sites (blue highlight) have been confirmed in other studies. Cyp19a1 has two E boxes (green highlight), HSD3B2 has an N box (red highlight), and Star has three E boxes and an N box. represses the Star promoter directly by binding to the HEY2-121 could inhibit GATA4-induced transcription E boxes or acts through a complex with GATA4, 3T3 cells (Fig. 7B). These data indicated that Hey proteins likely were transfected with the Star-luciferase reporter, regulate activation of steroidogenesis genes through plasmids expressing GATA4, and either full length specific sites in this promoter. HEY2 or one of two mutants (Fig. 7A). The first mutant, HEY2-62, lacks the N terminus, including the basic domain, but has an intact HLH domain. This mutant can Discussion form a dimer and interact with GATA4 but cannot bind Follicle maturation and growth includes an increase in DNA on its own (Kathiriya et al. 2004). The second the size of the oocyte and the proliferation and mutant, HEY2-121, lacks both the basic and HLH differentiation of granulosa cells, leading to antrum domains; it can neither dimerize nor bind DNA formation and increased steroidogenesis. Growth of the (Fig. 7A). When co-transfected with GATA4 and the follicle is regulated by the interplay of signals between Star-luciferase reporter gene, neither HEY2-62 nor the oocyte and the granulosa cells. A combination of www.reproduction-online.org Reproduction (2015) 150 383–394

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A 7000 oocyte factors, FSH, and estradiol are necessary for 6000 granulosa cell proliferation in preantral growing follicles (Moor et al. 1980, Robker & Richards 1998, Britt et al. 5000 2000, Kawashima et al. 2008, Murray et al. 2008, 4000 West-Farrell et al. 2009). Notch signaling has been 3000 shown to be important for granulosa cell proliferation. Disruption of Notch signaling in conditional mutants 2000 * of Notch2 and Jag1 in the ovary resulted in a lack of Relative luciferase activity 1000 * * granulosa cell proliferation (Zhang et al. 2011, Vanorny 0 et al. 2014). DAPT, a biomimetic inhibitor that is bound Star-luc + + + + + by the presenilin component of g-secretase, has been GATA4 – + + + + used to disrupt Notch signaling in vitro. This inhibitor HEY1 – – + – – preferentially targets amyloid precursor protein and HEY2 – – – + – Notch processing by g-secretase (Berezovska et al. HEYL – – – – + 2000, Hadland et al. 2001, Yang et al. 2008). Using DAPT to inhibit Notch signaling in ex vivo ovary culture B 5000 inhibited granulosa cell proliferation and folliculo- 4500 genesis was halted (Zhang et al. 2011, Manosalva et al. 4000 2013). In our studies, DAPT treatment resulted in the 3500 up-regulation of steroid biosynthesis genes in small 3000 follicles (Fig. 1). 2500 Based on our data, altered Notch signaling disrupts the 2000 normal process of steroid biosynthesis during folliculo- 1500 genesis, and this is consistent with the defective K/K Relative luciferase activity 1000 folliculogenesis noted in Lfng and conditional 500 * * mutants of Notch2, Jag1, and Hes1 (Hahn et al. 2005, 0 Manosalva et al. 2013, Xu & Gridley 2013, Vanorny HSD3B2-luc + + + + + et al. 2014). The data presented here demonstrates that GATA4 – + + + + Notch signaling has a role in the complex regulation of HEY1 – – + – – the expression of enzymes of the steroid biosynthesis HEY2 – – – + – pathway and likely regulates FSH-induced proliferation HEYL – – – – + of granulosa cells (Fig. 1). This is consistent with the role of Notch signaling in other tissues, promotion of C 300 proliferation, and regulation of the timing of the fully differentiated state. We propose that the differential 250 response to FSH, proliferation in small preantral follicles, 200 and increased steroidogenesis in larger preantral and antral follicles is regulated, in part, by Notch signaling. 150 In gonadal cells DAX1, a repressor, and the activator, * 100 SF1, regulate expression of multiple genes involved in steroidogenesis, including Cyp19a1 PII, Star, Cyp11A1, Relative luciferase activity 50 and HSD3B2 (Tremblay & Viger 2001a, Wang et al. 2001, 0 Lalli & Sassone-Corsi 2003, Jo & Stocco 2004, Manna Cyp19a1-luc + + + + + et al. 2009). Our studies indicated that activated Notch GATA4 – + + + + receptors can inhibit steroid synthesis in cAMP-activated HEY1 – – + – – MA-10 Leydig cells and in granulosa cells of preantral HEY2 – – – + – follicles (Figs 1 and 2), thus it was possible that expression HEYL – – – – + of either factor might be regulated by Notch. For example, Figure 6 Hey1, Hey2, and Heyl can inhibit promoters of steroid Dax1 transcription could be up-regulated by the Notch- biosynthesis genes. (A, B and C) NIH3T3 cells were transfected with ICD/RBPJK complex, blocking activation of SF1 and a luciferase reporter gene construct along with expression plasmids steroidogenesis. Alternatively, SF1 transcription could be for GATA4 and a HEY protein. Cell lysates were made 48 h post- inhibited by the HES or HEY repressors, also blocking transfection and luciferase activity determined. GATA4 activates each steroidogenesis. Treatment of MA-10 cells with dbcAMP luciferase reporter to high levels and this is significantly inhibited with specific Hey genes (*P!0.05, over bar). HEY1, HEY2, and HEYL results in a decrease in DAX1 protein levels, increasing differentially inhibit GATA4 mediated up-regulation of transcription the transcriptional activity of SF1 (Wang et al. 2002, of Star, Cyp19a1, and HSD3B1. All data are the results of three Rao et al. 2003, Jo & Stocco 2004, Trbovich et al. 2004, experiments done in triplicate GS.D. Manna et al. 2009). Our observations indicated that

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A that there was a differential inhibition of the promoters. bHLH O Y For example, the Star promoter was significantly HEY2 inhibited by HEY1, HEY2, and HEYL, but the HSD3B2 HEY2-62 promoter, which contains a single N box, was only inhibited by HEY1 and HEYL (Fig. 6). This is consistent HEY2-121 with previous data that demonstrated that HEY2 does not bind to N boxes with the sequence CACAAG (Nakagawa et al. 2000). Consistently, the Cyp19a1 promoter was not B 0 4500 inhibited by HEY2 or HEYL and the 5 most E box had a sequence that was not bound by HEY2 in previous 4000 studies (Nakagawa et al. 2000). The second E box found 3500 in this promoter is very similar in sequence and thus is likely a poor target for the HEY proteins also. These data 3000 indicate that direct DNA binding to specific sites in the 2500 promoters of these genes by the HEY proteins is likely the 2000 mechanism for Notch-mediated regulation of steroido- genesis gene transcription. We further ruled out the * 1500 likelihood of the HEY repressors acting through complex 1000 formation with GATA4 by using mutants of Hey2 (Fig. 7). Relative luciferase activity Our data demonstrated that GATA4-induced luciferase 500 activity was repressed only by full-length HEY2 and not 0 by a mutant that could form a complex with GATA4 but Star-luc + + + + + not bind DNA. In previous studies, there were no N or E GATA4 – + + + + boxes in the promoters examined, so this might indicate that in the absence of direct binding, the HEY repressors HEY2 – – + – – will act through complex formation with GATA factors HEY2-62 – – – + – (Kathiriya et al. 2004, Shirvani et al. 2006). HEY2-121 – – – – + Determining that the genes necessary for steroid biosynthesis in developing follicles and Leydig cells Figure 7 Hey proteins repress gene promoter activity through DNA binding. (A) The truncation mutants HEY2-62 (b) is missing the basic are Notch targets is a novel observation with important domain only, and HEY2-121 is missing both the basic and helix–loop– implications. Notch signaling is important for the helix domains (bHLH). (B) NIH3T3 cells were transfected with the Star- development of the male reproductive tract, and Leydig luciferase reporter along with plasmids that express GATA4 and HEY2, cells particularly (Dirami et al. 2001, Tang et al. 2008, HEY2-62, or HEY2-121. HEY2 significantly inhibited GATA4-induced Hahn et al. 2009, Defalco et al. 2013). Perturbation of luciferase activity. The loss of either the basic or bHLH domains of Notch signaling in granulosa cells results in MOFs, Hey2 resulted in a loss of repression of the Star-luciferase reporter gene (*P!0.05) and indicated that DNA binding was necessary for HEY2- defective meiotic maturation, and loss of oocytes (Hahn mediated inhibition of this promoter. All data are the results of three et al. 2005, Trombly et al. 2008, Manosalva et al. 2013, experiments done in triplicate GS.D. Xu & Gridley 2013, Vanorny et al. 2014). Because Notch2, Notch3, and Jag2 are not expressed in the oocyte, it is logical to conclude that these defects arise Notch-mediated inhibition of steroid biosynthesis was because altered Notch signaling disrupted the reciprocal not achieved by modulating the expression of these two communication between the oocyte and granulosa cells. major transcriptional regulators (Fig. 3) but rather by Also, GATA factors have important roles in gonad repressing expression of genes that encode important development and the function and regulation of steroid nodal points in gonadal steroid biosynthesis. biosynthetic genes (Viger et al. 2008, Padua et al. 2014, Steroidogenesis genes have well-studied promoters 2015, Tevosian 2014, Bergeron et al. 2015, Schrade that are transactivated by GATA4 (Tremblay & Viger et al. 2015). Conditional alleles of Gata4 resulted in 2001a,b, Tremblay et al. 2002, Viger et al. 2004, 2008, impaired fertility in both adult female and male mice, K K Bergeron et al. 2015, Schrade et al. 2015). All three Gata4 / ovaries ovulated fewer oocytes and developed activated Notch receptors significantly inhibited ovarian cysts, and there was decreased Cyp19a1 HSD3B2, Star, and Cyp19a1 promoter activity (Fig. 4). expression (Kyro¨nlahti et al. 2011a). When Gata4 and This observation is consistent with work by others that Gata6 are simultaneously deleted in granulosa cells, showed that the transactivation ability of GATA4 can be there is a complete lack of proliferation and no follicular inhibited by Notch through HEY2 (Kathiriya et al. 2004). development in the adult (Padua et al. 2014). Knock- We determined that the Cyp19a1 PII, HSD3B2, and Star down or deletion of Gata4 in Leydig cells resulted in promoters had E and N boxes that HEY repressors could decreased expression of genes involved in cholesterol bind (Fig. 5), and subsequent experiments demonstrated synthesis and transport and steroid biosynthesis www.reproduction-online.org Reproduction (2015) 150 383–394

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(Bergeron et al. 2015, Schrade et al. 2015). The homolog-1, regulates human aromatase (CYP19) PII promoter activity in disruption of GATA4 function or conditional loss of breast cancer cells. Endocrinology 146 4905–4916. (doi:10.1210/en. 2005-0187) Gata4 in Sertoli cells in adult males also resulted in a loss Britt KL, Drummond AE, Cox VA, Dyson M, Wreford NG, Jones MEE, of fertility and repression of steroid biosynthesis genes Simpson ER & Findlay J 2000 An age-related ovarian phenotype in mice (Kyro¨nlahti et al. 2011b, Schrade et al. 2014, Bergeron with targeted disruption of Cyp 19 (Aromatase) gene. Endocrinology 141 et al. 2015, Padua et al. 2015). HEY2 has been 2614–2623. (doi:10.1210/endo.141.7.7578) Chin MT, Maemura K, Fukumoto S, Jain MK, Layne MD, Watanabe M, demonstrated to be an important factor in the regulation Hsieh CM & Lee ME 2000 Cardiovascular basic helix loop helix factor 1, of GATA-mediated transactivation of cardiac specific a novel transcriptional repressor expressed preferentially in the genes (Kathiriya et al. 2004, Fischer et al. 2005), and our developing and adult cardiovascular system. Journal of Biological Chemistry 275 6381–6387. (doi:10.1074/jbc.275.9.6381) data implicates the HEY proteins in the regulation of Cortvrindt R, Smitz J & Van Steirteghem AC 1997 Assessment of the steroidogenesis postnatally. Because Notch genes are need for follicle stimulating hormone in early preantral mouse follicle expressed in adrenal glands (de Mendonca et al. 2014) culture in vitro. Human Reproduction 12 759–768. (doi:10.1093/ and the developing pituitary (Goldberg et al. 2011), the humrep/12.4.759) Defalco T, Saraswathula A, Briot A, Iruela-Arispe ML & Capel B 2013 possibility that alterations to the Notch pathway can Testosterone levels influence mouse fetal Leydig cell progenitors through affect steroidogenesis and potentially the hypothalamic– Notch signaling. Biology of Reproduction 88 91. (doi:10.1095/ pituitary–gonadal axis will be an important future biolreprod.112.106138) consideration. Dirami G, Ravindranath N, Achi MV & Dym M 2001 Expression of Notch pathway components in spermatogonia and Sertoli cells of neonatal mice. Journal of Andrology 22 944–952. (doi:10.1002/j1939-4640. 2001.tb03434.x) Declaration of interest Elagib KE, Xiao M, Hussaini IM, Delehanty LL, Palmer LA, Racke FK, The authors declare that there is no conflict of interest that Birrer MJ, Shanmugasundaram G, McDevitt MA & Goldfarb AN 2004 Jun blockade of erythropoiesis: role for repression of GATA-1 by HERP2. could be perceived as prejudicing the impartiality of the Molecular and Cellular Biology 24 7779–7794. (doi:10.1128/MCB.24. research reported. 17.7779-7794.2004) Fischer A, Klattig J, Kneitz B, Diez H, Maier M, Holtmann B, Englert C & Gessler M 2005 Hey basic helix–loop–helix transcription factors are Funding repressors of GATA4 and GATA6 and restrict expression of the GATA target gene ANF in fetal hearts. Molecular and Cellular Biology 25 This work was funded in part by NICHD grant HD39850-01 to J 8960–8970. (doi:10.1128/MCB.25.20.8960-8970.2005) Wilson-Rawls. 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Revised manuscript received 9 July 2015 Endocrinology 142 977–986. (doi:10.1210/endo.142.3.7995) Accepted 16 July 2015

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